Abstract: Base on the internal transcribed spacers (ITS) sequences of Trichoderma spp.,a pairs of specificity primers(DG and DT) were designed for the real-time quantitative PCR assay of soil Trichoderma. The optimal reaction system of real-time quantitative PCR was established. The masses of genomic DNA of Trichoderma spp. in soybean filed were quantified by using this reaction system,and the genomic DNA was extracted from three long-term fertilization management; no fertilization(NF),chemical fertilization applied with nitrogen and phosphorus(NP) and chemical N,P combined with pig manure(NPM). Primers DG and DT possessed a good specificity to the amplification of Trichoderma spp..The correlation coefficient(R²) and slope of the equation from the standard curve were 0.993 and -0.282 9 respectively,and the melting curve only showed a single peak. At seedling stage of soybean growth stage,the mass of genomic DNA of Trichoderma spp. in NF,NP and NPM treatments were 0.4 ng,0.8 ng and 1.2 ng per gram of soil,respectively. The mass in NPM measure was higher than that in NF and NP measure significantly (P<0.05).